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PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :
G01N 27/327

(11) International Publication Number: WO 97/02487

(43) International Publication Date: 23 January 1997 (23.01.97)

(21) International Application Number: PCT/US9671 1 240

(22) International Filing Date: 28 June 1996 (28.06.96)

(30) Priority Data:

08/496.939

30 June 1995 (30.06.95)

(71) Applicant: BOEHRINGER MANNHEIM CORPORATION

[US/US]; 91 15 Hague Road, P.O. Box 50528, Indianapolis,
IN 46250 (US).

(72) Inventors: PRITCHARD, G., John; 59 Wfldrose Drive, An-

cover, MA 01810 (US). BATESON, Joseph, E4 14817
Senator Way, Carmel, IN 46032 (US). HILL, Brian, S.;
Apartment C, 4710 San Fernando Drive, Indianapolis, IN
46268 (US). HEALD, Brian, A.; 10337 Seagrave Drive,
Fishers, IN 46038 (US). HUBBARD, Scott, E.; 1261 W.
Furry Road, Fountaintown, IN 46130 (US).

(74) Agents: YOUNG, D., Michael et al.; Boehringer Mannheim
Corporation, 9115 Hague Road, P.O. Box 50528, Indianapo-
lis, IN 46250 (US).

(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY.
CA, CH, CN, CZ, DE, DK, EE, ES, FI. GB, GE, HU, IS,
JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD,
MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD,
SE, SG. SI, SK, TJ, TM, TR, IT, UA, UG. UZ, VN, ARIPO
patent (KE, LS, MW, SD, SZ, UG), Eurasian patent (AM,
AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT,
BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC,
NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA,
GN, ML, MR, NE, SN, TD, TG).

Published

With international search report.

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of
amendments.

(54) Title: ELECTROCHEMICAL BIOSENSOR TEST STRIP

(57) Abstract

An electrochemical biosensor test strip (1) mat has a minimum volume blood sample requirement of about 9 microliters. The test
strip has working (4) and counter (5) electrodes that are substantially the same size and made of the same electrically conducting material
placed on a first insulating substrate (2). Overlaying the electrodes is a second insulating substrate (3), which includes a cutout portion (8)
that forms a reagent well. The cutout portion exposes a smaller area of the counter electrode than the working electrode. A reagent for
analysis of an analyte substantially covers the exposed areas of working and counter electrodes in the reagent well. Overlaying the reagent
well and affixed to the second insulating substrate is a spreading mesh (13) that is impregnated with a surfactant The small cutout portion
of 4 millimeters by 4.2 millimeters, small mesh of 6 millimeters by 5.8 millimeters, and small amount of reagent, 4 microliters before
drying, allow the test strap to analyze a whole blood sample of about 9 microliters.

FOR TUB PURPOSES OP INFORMATION ONLY

Codes used to identify States party to the POT on the front pages of pamphlets publishing international
applications under the PCT.

Armaria

Austria

^^Kmgdora

Australia

- Guinea

Barbados

Greece

Belgium

Hungary

Btttin Faso

Ireland

Bulgaria

Italy

Benin

Japan

Brazil

Kenya

Belarus

Kyrgysfan

Canada

Democratic People's Republic

Central African Republic

of Korea

Congo

Republic of Korea

Switzerland

Coted'tvoire

LfeducmlciB

Cameroon

Sri Lanka

China

Lmeria

Czechoslovakia

Luxembourg

Germany

Latvia

Denmark

Monaco

Republic of Moldova

Spam

Madagascar

UG
US

Finland

Mali

Mongolia

Gabon

Mauritania

Mexico
Niger

Norway
New Zealand

Russian Federation

Sweden

Singapore

Slovenia

Senegal

Togo
Tajikistan

Trimdad and Tobago

Ukraine

Uganda

United States of America

Uzbekistan

Viet Nam

WO 97/02487 PCT/US96/11240
ELECTROCHEMICAL BIOSENSOR TEST STRIP
Cross-Rcferences to Related Application^

5 This application is a continuation-in-part of U.S. patent application no.

08/198,407, filed February 21, 1994, the disclosure of which is hereby incorporated
by reference.

Field of The Invention

This invention relates generally to the determination of the concentration of
analytes in fluids and more specifically to an amperometric biosensor for use in such
determination.

15 Background of The Invention

Biosensors are not new. Their use in the determination of concentrations of
various analytes in fluids is also known.

20 Nankai et al., WO 86/07632, published December 31, 1986, discloses an

amperometric biosensor system in which a fluid containing glucose is contacted with
glucose oxidase and potassium ferricyanide. The glucose is oxidized and the
ferricyanide is reduced to ferrocyanide. (This reaction is catalyzed by glucose
oxidase.) After two minutes, an electrical potential is applied and a current caused

25 by the re-oxidation of the ferrocyanide to ferricyanide is obtained. The current
value, obtained a few seconds after the potential is applied, correlates to the
concentration of glucose in the fluid.

Because Nankai et al. discloses a method in which the reaction of glucose and
3 0 ferricyanide may run to completion prior to the application of an electrical potential,
this method is referred to as the "end-point" method of amperometric determination.

Nankai et al. discloses a system, wherein the glucose oxidase and potassium
ferricyanide are held on a nonwoven nylon mesh. The mesh is positioned so that it is
3 5 in contact with a working electrode, a counter electrode and a reference electrode.

W ° 97/02487 PCT/US-K/1,240

The total surface area of the counter and reference electrodes is twice that of the
working electrode.

Wogoman, EP 0 206 218, published Dec. 30, 1986 discloses a biosensor
5 having two electrodes, the electrodes being made of different electrically conducting
materials. For example, the anode is formed from an anode material, such as
platinum, and the cathode is formed from a cathode material, such as silver. The
anode is coated with an enzyme. In a preferred embodiment, the coated electrode is
covered with an elastomer that is permeable to glucose.

Pottgen et al., WO 89/08713, published Sept. 21 , 1989, discloses the use of a
two electrode biosensor, wherein the electrodes are made of the same noble metal,
but one of the electrodes (referred to as a pseudoreference electrode) is larger than
the other (working) electrode.

Recently, Pollmann et al., U.S. Patent No. 5,288,636, issued Feb. 22, 1994,
disclosed an electrochemical biosensor test strip that includes working and counter
electrodes of substantially the same size and made of the same electrically conducting
materials. The Pollmann et al. test strip includes a reagent well that will

20 accommodate a testing sample of human whole blood from about 10 to about 70
microliters. However, below about 13 microliters, errors in the measurement of an
analyte, such as glucose, from a whole blood sample may result (low dosing errors).
Generally, the low dosing error is manifested as an understated measurement of the
analyte, or no measurement of the analyte by the meter used in conjunction with the

25 test strip. Low dosing errors are a particular concern for infants and elderly persons
who often have difficulty in expressing a reasonably sized blood drop for testing
upon pricking their finger with a lancet.

Accordingly, it is highly desirable to design a test strip that requires a
30 niinimum volume of blood for the testing of an analyte, such as blood glucose.

Summary of the Invention

WO 97/02487 PCT/US96/11240

The invention is an electrochemical biosensor test strip that has a lower
minimum volume blood sample requirement than prior art strips of similar
construction. The present inventive test strip has a smaller reagent well and smaller
spreading mesh than similar prior art strips. Further, the reagent well is positioned
5 differently than in similar prior art test strips. The minimum blood volume sample
requirement for the new strip is about 9 microliters.

The smaller sample volume requirement means fewer low sample volume
dosing errors result when measuring an analyte, such as glucose, from a whole blood

10 sample. This result is especially important for those persons, such as infants and the
elderly, who have difficulty expressing a reasonably sized drop of blood by pricking
their finger with a lancet. Also, with the present inventive strip it is easier for the
meter, which collects current measurements and correlates those measurements to a
concentration of analyte from a sample, to discriminate low sample volume dosing

15 errors. Further, the smaller reagent well requires less reagent per biosensor strip,
thereby increasing the production volume for mass production of biosensor test
strips.

Additionally, when the spreading mesh is affixed to the test strip by an
20 adhesive tape, the tape includes a hole that exposes the reagent well and spreading
mesh, and further includes air vents on opposing sides of the hole. These air vents
reduce the occurrence of air bubbles trapped in the reagent well when a sample is
being tested. Air bubbles can produce testing errors.

25 Brief Description of the Drawings

FIG. 1 is an exploded view of the present inventive biosensor test strip.

FIG. 2 is a top view of the biosensor test strip without the reagent, spreading
3 0 mesh, and adhesive tape with air vents.

FIG. 3 is a top view of the fully constructed, preferred biosensor test strip.

FIG. 4 is a cross-sectional view of the biosensor of FIG. 3 along lines 21-21 .

FIG. 5 illustrates hypothetical calibration curves for different lots of

WO 97/02487 PCT/US96/11240

biosensor test strips.
Description of the Preferred Embodiment

5 The present inventive biosensor test strip is similar to the preferred

embodiment of the test strip described in Pollmann et al., U.S. Patent No. 5,288,636,
issued Feb. 22, 1994, the disclosure of which is hereby incorporated by reference.
However, the Pollmann et al. strip has a construction such that too many low dosing
errors result when whole blood samples below about 13 microliters are tested for
10 blood glucose.

In the present inventive test strip, reagent well 2 (Fig. 4) has been reduced in
size over the Pollmann et al. reagent well and repositioned so that a smaller surface
area of the counter electrode 5 than the working electrode ± is exposed by cutout
15 portion S, which forms reagent well 2. (Figs. 1-4) Mesh 12, which is a spreading
mesh, is also reduced in size over the Pollmann et al. mesh. (Figs. 1, 3, 4) These
changes in strip architecture result in a test strip that can accurately measure an
analyte, such as glucose, from a minimum whole blood sample of about 9
microliters.

Referring specifically to Figs. 1 through 4, there is shown the presently
preferred embodiment of the inventive biosensor test strip.

Test strip I comprises first and second electrically insulating layers 2 and 2,
25 respectively. Any useful insulating material will be suitable. Typically, plastics,
such as vinyl polymers and polyimides provide the electrical and structural properties
which are desired. Preferably, these layers are Melinex 329, 7 mil.

The biosensor test strip shown in Figs. 1 through 4 is intended to be mass
3 0 produced from rolls of material, necessitating the selection of a material which is
sufficiently flexible for roll processing and at the same time sufficiently stiff to give a
useful stiffiiess to the finished biosensor test strip.

WO 97/02487

Layers 2 and 2 may be of any usefiil thickness,
layers 2 and 2 are about 7 mil thick.

PCT/US96/11240
In a preferred embodiment,

Working electrode 4 and counter electrode 5 are preferably deposited on a
5 backing of insulator material 2, such as polyimide, to reduce the possibility of tearing
the electrode before it is affixed to layer 2. Working electrode 4 and counter
electrode 5 are substantially the same size and are made of the same electrically
conducting material. Examples of electrically conducting materials that may be used
are palladium, platinum, gold, silver, carbon, titanium, and copper. Noble metals

10 are preferred because they provide a more constant, reproducible electrode surface
area. Palladium is particularly preferred because it is one of the more difficult noble
metals to oxidize and because it is a relatively inexpensive noble metal. Silver is not
preferred because it is more readily oxidized by air than the other noble metals listed
above. Preferably, electrodes 4 and 5 are about 0. 1 micron thick and backing 2 is

15 about 25 microns thick (commercially available from Courtaulds Performance Films
in California and Southwall Technologies, Inc.).

Electrodes 4 and 5 must be sufficiently separated so that the electrochemical
events at one electrode do not interfere with the electrochemical events at the other
20 electrode. The preferred distance between electrodes 4 and 5 is about 1.2
millimeters.

In the preferred embodiment, electrodes 4 and 5, affixed to backing 2, are
unspooled from reels and attached to layer 2 by the use of hot melt adhesive (not
25 shown). Electrodes 4 and 5 also preferably extend from one end of layer 2 to the
other end in parallel configuration.

Insulating layer 2 is fixed on top of layer 2 and electrodes 4 and.5 by the use
of hot melt adhesive (not shown). Layer 2 includes cutout portion 3, which defines
3 0 reagent well 2. Both the size and the position of cutout portion S are critical to the
invention. Cutout portion £ must be sufficiently small and must be sufficiently
positioned such that in combination with the spreading mesh, described below, a

WO 97/02487 PCT/US96/11240

minimum whole blood sample volume of about 9 microliters may be accurately
analyzed by the test strip. The preferred size of cutout portion S is 4 millimeters by
4.2 millimeters.

5 In the preferred embodiment, the 4 mm side of cutout portion S runs parallel

to the long side of the test strip shown in Figs. 1-4. Importantly, cutout portion 8 is
positioned over electrodes 4 and 5 such that a smaller surface area of counter
electrode 5 than working electrode 4 is exposed. Preferably, the exposed surface
area of working electrode 4 is twice as large as the exposed surface area of counter
10 electrode 5. Surprisingly, offsetting cutout portion £ to expose a smaller surface area
for the counter electrode than the working electrode does not adversely affect
measurement of an analyte from a sample being measured. In this preferred
embodiment, electrodes 4 and 5 are 1.5 mm in width.

15 Biosensor test strip 1 may be accompanied by a power source (not shown) in a

electrical connection with the working and counter electrodes and a current
measuring meter (not shown) which is also in a electrical connection with the
working and counter electrodes.

20 Biosensor reagent 11 (Fig. 4) is placed in well 2 so that it covers substantially

all of exposed surfaces 1Q and 2Q of working electrode 4 and counter 5, respectively.
(Figs. 2-4) An example of a reagent that may be used in the biosensor test strip of
the present invention is a reagent for measuring glucose from a whole blood sample.

25 A protocol for making a glucose reagent utilizing the enzyme glucose oxidase

and ferricyanide as the oxidized form of the redox mediator is as follows:

Step 1- Prepare 1 liter (in a volumetric flask) of a buffer/NATROSOL mixture by
adding 1.2000 grams (g) NATROSOL-250 M to 0.740 M aqueous potassium
30 phosphate buffer (including 80.062 g monobasic potassium phosphate and 26.423 g
dibasic potassium phosphate) at pH 6.25. Allow the NATROSOL to stir and swell
for 3 hours.

WO 97/02487

PCT/US96/11240

Step 2- Prepare an AVICEL mixture by stirring 14.0000 g AVICEL RC-591 F and
504.7750 g water for 20 minutes.

5 Step 3- Prepare a TRITON mixture by adding 0.5000 g TRITON X-100 to
514.6000 g of the buffer/NATROSOL mixture and stir for 15 minutes.

Step 4- While stirring, add the total TRITON mixture dropwise with an addition
funnel or buret to the total AVICEL mixture. Once addition is complete, continue
10 stirring overnight.

Step 5- To the mixture resulting from Step 4, add, while stirring, 98.7750 g
potassium ferricyanide. (Add a little potassium ferricyanide at a time to allow the
potassium ferricyanide to dissolve as added.)

Step 6- Stir the resulting mixture of Step 5 for 20 minutes.

Step 7- Adjust the pH of the mixture resulting from Step 6 to 6.25 by adding
potassium hydroxide.

Step 8- To the resulting mixture of Step 7, add 9. 1533 g glucose oxidase (218.50
tetramethyl benzidine units per milligram (mg) from Biozyme) and stir at least 20
minutes.

25 Step 9- To the resulting mixture of Step 8, add 20 g potassium glutamate and stir
at least 20 minutes. ; \

Step 10- Filter the resulting mixture of Step 9 through a 100 micron sieve bag to
remove any AVICEL clumping. The filtrate is the resulting reagent composition
30 (reagent 11), which is added to reagent well 2 and is then dried at about 50 C° for
about 3 minutes.

WO 97/024*7

PCT/US96/11240

In the preferred embodiment for glucose determination, 4 microliters of
reagent made by the above-stated protocol is added to well 2 formed by cutout 2.
This amount of reagent H will substantially cover surface areas ifl and 2Q of the
5 electrodes 4 and 5 (Fig, 2) and will also contain a sufficient amount of ferricyanide,
and a sufficient amount of enzyme (glucose oxidase) to catalyze the oxidation of
glucose (from a sample of human whole blood) and the reduction of ferricyanide to
completion, as defined herein, within about 20 seconds. (Prior to adding the reagent
to well 2, it is preferable to treat well 2 with a 600 Watt corona arc, gapped at
l o 1/40,000 inch on a processing line travelling at 4 meters per minute, to make well 9
more hydrophilic, thereby allowing the reagent to spread more evenly in the well.)

Another glucose reagent that may be formulated includes 300 millimolar
potassium ferricyanide, 250 millimolar potassium phosphate buffer, 14 grams

15 microcrystalline cellulose (AVICEL RC-591 F) per liter of reagent, 0.6 grams

hydroxyethylcellulose (NATROSOL-250 M) per liter of reagent, 0.5 grams Triton X-
100 surfactant per liter of reagent, 37 millimolar sodium succinate, and 1.57 million
tetramethyl benzidine units of glucose oxidase per liter of reagent. Sodium hydroxide
(6 Normal solution) is used to titrate this reagent to a pH of 6.6. This reagent may

20 formulated by the same protocol described above, but amounts of components
should be adjusted and components substituted (sodium succinate for potassium
glutamate and sodium hydroxide for potassium hydroxide) to achieve the component
concentrations stated above. Drying of this reagent in reagent well 2 typically results
in a loss of enzyme activity of about 30-35 % .

After drying reagent 11, a spreading mesh 12, which has been impregnated
with a surfactant, is placed over cutout portion 3 and is affixed to second electrical
insulator 2. Speading mesh 12 is preferably a polyester monofilament mesh from
ZBF (Zurich Bolting Cloth Mfg. Co. Ltd., Ruschlikon, Switzerland). The spreading
3 0 mesh is preferably dipped in a solution of 0.8% (wt. :vol.) dioctylsodium

sulfosuccinate (DONS) in a solution of 50:50 (vol.: vol.) methanol: water, and then
dried. Spreading mesh 13 must be small enough such that in combination with the

WO 97/02487 PCT/US96/11240

size of cutout portion 3 and placement of cutout portion fi the biosensor strip will
accurately measure analyte from a minimum whole blood sample of about 9
microliters. The preferable dimensions of spreading mesh 12 are 6 mm x 5.8 mm.
In the most preferred biosensor strip, the 6 mm side of the mesh is parallel to the
5 long side of the strip shown in Figs. 1-4.

Preferably, spreading mesh 12 is affixed to adhesive tape 14, which includes
hole 15. (Figs. 1, 3, 4) Adhesive tape 14 is preferably made of polyester with an
adhesive backing. (Available from Tapemark, Medical Products Division, 223 E.

10 Marie Ave., St. Paul, Minnesota 55118) Adhesive tape 14 is preferably dyed

maroon and hole 15 provides a target area for application of a sample to be analyzed
by the biosensor. Hole 15 exposes at least a portion of spreading mesh 12 and cutout
portion §, and preferably exposes substantially all of cutout portion 8. Tape 14
preferably includes slits 16, as shown in Figs. 1 and 3, located on opposing sides of

15 hole 15. (Two slits 1£ are shown in Figs. 1 and 3, but one slit may be sufficient.)
Slits 1£ constitute air vents, which reduce the occurrence of air bubbles trapped in
the reagent well upon the addition of a sample such whole blood to the reagent well.
Reducing the occurrence of air bubbles trapped in reagent well 2 results in fewer
testing errors.

After drying the reagent and affixing the spreading mesh, the roll-formed
biosensors are separated by die punching to form discrete biosensors, which are used
in conjunction with 1) a power source in electrical connection with the working and
counter electrodes and capable of supplying an electrical potential difference between

25 the working and counter electrodes sufficient to cause diffusion limited

electrooxidation of the reduced form of the redox mediator at the surface of the
working electrode, and 2) a meter in electrical connection with the working and
counter electrodes and capable of measuring the diffusion limited current produced by
oxidation of the reduced form of the redox mediator when the above-stated electrical

30 potential difference is applied.

WO 97/02487 PCT/US96/11240
The meter described above will normally be adapted to apply an algorithm
(discussed below) to the current measurement, whereby an analyte concentration is
provided and visually displayed. Improvements in such power source, meter, and
biosensor system are the subject of commonly assigned U.S. Patent Number
5 4,963,814, issued October 16, 1990; U.S. Patent No. 4,999,632, issued March 12,
1991; U.S. Patent No. 4,999,582, issued March 12, 1991; U.S. Patent No.
5,243,516, issued September 7, 1993; U.S. Patent No. 5,352,351, issued Oct. 4,
1994; U.S. Patent No. 5,366,609, issued Nov. 22, 1994; White et al., U.S. Patent
Application Serial No. 08/073,179, filed 6/8/93 (Issue Fee mailed 12/27/94); and
10 White et al., U.S. Patent Application Serial No. 08/343,363, filed 11/22/94 (Issue
Fee mailed 5/5/95), the disclosures of which are hereby incorporated by reference.

For easy electrical connection of the power source and meter, additional
cutout portion 12 (Figs. 1 through 4), exposing portions of the working and counter
15 electrodes, are preferably provided in the biosensor device.

The biosensor device described above may be used to determine the
concentration of an analyte in a fluid sample by performing the following steps:

20 a) contacting a fluid sample, such as whole blood, with a reagent

(described above) that substantially covers surface areas Ifl and 2Q of working
and counter electrodes 4 and 5, respectively;

b) allowing the reaction between the analyte and the oxidized form of the
25 redox mediator to go to completion, as defined herein;

c) subsequently applying a potential difference between the electrodes
sufficient to cause diffusion limited electrooxidation of the reduced form of
the redox mediator at the surface of the working electrode;

d) thereafter measuring the resulting diffusion limited current; and

WO 97/02487 PCT/DS96/11240

e) correlating the current measurement to the concentration of analyte in
the fluid. (Reaction completion is defined as sufficient reaction between the
analyte and the oxidized form of the redox mediator to correlate analyte
concentration to diffusion limited current generated by oxidation of the
5 reduced form of the redox mediator at the surface of the working electrode.)

Many analyte-containing fluids may be analyzed. For example, analytes in
human body fluids such as whole blood, blood serum, urine and cerebrospinal fluid
may be measured. Also, analytes found in fermentation products and in
10 environmental substances, which potentially contain environmental contaminants,
may be measured.

When measuring analytes found in human body fluids, especially whole
blood, the potential difference applied between the electrodes is preferably no more

!5 than about 500 millivolts. When a potential difference above about 500 millivolts is
applied between the electrodes, oxidation of the working electrode surface (for
palladium) and of some blood components may become intolerable, thereby
preventing an accurate arid precise correlation of current to analyte concentration.
For an assay of glucose in a whole blood sample, wherein the oxidized form of the

20 redox mediator is ferricyanide, a potential difference from about 150 millivolts to
about 500 millivolts may be applied between the electrodes to achieve diffusion
limited electrooxidation of the reduced form of the redox mediator at the surface of
the working electrode. Preferably, about 300 millivolts potential difference is applied
between the electrodes.

Current generated from the oxidation of the reduced form of the redox
mediator may be measured at any time from about 0.5 seconds to about 30 seconds
after the potential difference is applied between the electrodes. At less than about 0.5
seconds, diffusion limited current is difficult to measure due to the charging current.
30 After about 30 seconds, convection becomes significant, thereby interfering with the
measurement of a diffusion limited current.

WO 97/02487 PCT/US96/11240

The current measured during the assay of an analyte from a fluid sample may
be correlated to concentration of the analyte in the sample by application of an
algorithm by the current measuring meter. The algorithm may be a simple one, as
illustrated by the following example:

[Analyte] = Ci 7,5 + d

wherein [Analyte] represents the concentration of the analyte in the sample (see Fig.
5), i 7 5 is the current (in microamps) measured at 7.5 seconds after application of
10 the potential difference applied between the electrodes, C is die slope of line 22 (Fig.
5), and d is the axis intercept (Fig. 5).

By making measurements with known concentrations of analyte, calibration
curve 22 (Fig. 5) may be constructed. This calibration will be stored in the Read
15 Only Memory (ROM) key of the meter and will be applicable to a particular lot of
biosensor test strips. Lines 24 and 26 in Fig. 5 represent other hypothetical
calibration curves for two other different lots of biosensor test strips. Calibration for
these biosensor lots would generate slightly different values for C and d in the above
algorithm.

In analysis of glucose from a sample of human whole blood, 20 *il of whole
blood is preferably added to the above-stated glucose reagent. The reaction of
glucose and ferricyanide is allowed to go to completion, thereby forming gluconic
acid and ferrocyanide. This reaction normally requires a short time, preferably less

25 than about 20 seconds, to go to completion. About twenty seconds after addition of
the whole blood sample, a potential difference of about 300 millivolts is applied
between the electrodes, thereby oxidizing ferrocyanide to ferricyanide at the surface
of the working electrode. Current measurements are made at 0.5 second intervals
from 1 second to 7.5 seconds after the potential difference is applied between the

30 electrodes. These current measurements are correlated to the concentration of
glucose in the blood sample.

WO 97/02487

PCIYUS96/11240

In this example of measuring glucose from a blood sample, current
measurements are made at different times (from 1 second to 7.5 seconds after
application of the potential difference), rather than at a single fixed time (as described
5 above), and the resulting algorithm is more complex and may be represented by the
following equation:

[Glucose] = Ci ii + C 2 i 2 + C 3 i 3 + ...C n i n + d, wherein i\ is the
current measured at the first measurement time (1 second after application of the 300

10 millivolt potential difference), i 2 is the current measured at the second measurement
time (1.5 seconds after application of the 300 millivolt potential difference), i 3 is the
current measured at the third measurement time (2 seconds after application of the
300 millivolt potential difference), i n is the current measured at the n* measurement
time (in this example, at the 14 th measurement time or 7.5 seconds after application

15 of the 300 millivolt potential difference), Cj, C 2 , C3, and C n are coefficients derived
from a multivariate regression analysis technique, such as Principle Components
Analysis or Partial Least Squares, and d is the regression intercept (in glucose
concentration units). (A modification of this procedure may be used in the event that
calibration curves illustrated by Fig. 5 have considerable curvature.)

Alternatively, the concentration of glucose in the sample being measured may
be determined by integrating the curve generated by plotting current, i, versus
measurement time over some time interval (for example, from 1 second to 7.5
seconds after application of the 300 millivolt potential difference), thereby obtaining
25 the total charge transferred during the measurement period. The total charge
transferred is directly proportional to the concentration of glucose in the sample
being measured.

Further, the glucose concentration measurement may be corrected for
30 differences between environmental temperature at the time of actual measurement
and the environmental temperature at the time calibration was performed. For
example, if the calibration curve for glucose measurement was constructed at an

WO 97/02487

PCT/US96/11240

environmental temperature of 23°C, the glucose measurement is corrected by using
the following equation:

[Glucose] corrected = [Glucose] measured * (1-K(T-23°C)), wherein T is the
5 environmental temperature (in °C) at the time of the sample measurement and K is a
constant derived from the following regression equation:

Y = K(T-23),

10 wherein Y = [Glucose] measured at 23 °C ' ^ lucose lmeasured at T°C

[Gl«cose] measuredatTOC

In order to calculate the value of K, each of a multiplicity of glucose concentrations
15 is measured by the meter at various temperatures; T, and at 23°C (the base case).
Next, a linear regression of Y on T-23 is performed. The value of K is the slope of
this regression.

The glucose concentration of a sample may be accurately and precisely
20 measured by the present inventive method utilizing the present inventive biosensor.
Further, when a sample of human whole blood is measured, error due to hematocrit
effect is insignificant in the range of 30-55% hematocrit.

Other examples of enzymes and redox mediators (oxidized form) that may be used in
25 measuring particular analytes by the present invention are listed below in Table 1.

TABLE 1

ANALYTE

REDOX MEDIATOR
(OXIDIZED FORM)

ADDITIONAL MEDIATOR

GLUCOSE

GLUCOSE DEHYDROGENASE
AND DIAPHORASE

FERRICYANIDE

GLUCOSE

GLUCOSE-DEHYDROGENASE
(QUINOPROTEIN)

FERRICYANIDE

CHOLESTEROL

CHOLESTEROL ESTERASE
AND CHOLESTEROL OXIDASE

FERRICYANIDE

2,6-DIMETHYL-M-

BENZOQUINONE

2 T 5-DICHLORO-J,4-

BENZOQUINONE

OR PHENAZINE

ETHOSULFATE

WO 97/02487

PCT/USSW11240

HDL

L.JrlUJLco 1 fcKUL

CHOLESTEROL ESTERASE
AND CHOLESTEROL OXIDASE

FERRICYANIDE

2,6-DIMETHYI^l,4-

BENZOQUINONE

2,5-DICHL0RO-l,4-

BENZOQUINONE

OR PHENAZINE

ETHOSULFATE

1 KJ VjL, I LmiUcj

LtPUFKU I EIN LIPASE,
GLYCEROL KINASE, AND
GLYCEROL-3-PHOSPHATE
OXIDASE

FEkRICYANIDE OR

PHENAZINE

ETHOSULFATE

PHENAZINE
METHOSULFATE

LACTATE

LACTATE OXIDASE

FERRICYANIDE

2.6-DICHLORO-1.4-
BENZOQUINONE

l*r\ v. i t\ 1 C

LAv lAlt UxM I UKUubN ASE
AND DIAPHORASE

FERRICYANIDE,

PHENAZINE

fc I HUSULr ATE, OR

PHENAZINE

M ETHOSULFATE

DEHYDROGENASE

rfcJUUCYANIDE,
PHENAZINE
ETHOSULFATE, OR
PHENAZINE
METHOSULFATE

PYRUVATE

PYRUVATE OXIDASE

FERRICYANIDE

ALCOHOL

ALCOHOL OXIDASE

PHENYLENEDIAMINE

BILIRUBIN

BILIRUBIN OXIDASE

I-METHOXY-

PHENAZINE

METHOSULFATE

URIC ACID

URICASE

FERRICYANIDE

In some of the examples shown in Table 1 , at least one additional enzyme is
used as a reaction catalyst. Also, some of the examples shown in Table 1 may utilize
an additional mediator, which facilitates electron transfer to the oxidized form of the
5 redox mediator. The additional mediator may be provided to the reagent in lesser
amount than the oxidized form of the redox mediator.

When compared to the preferred embodiment of the closest prior art biosensor
test strip, disclosed in Pollmann et al., the present inventive biosensor has the
10 following distinguishing features:

1 . reagent well 9 is 30% smaller;

2. when the working and counter electrodes are substantially the same
15 size, the exposed surface area of the counter electrode in the reagent well is less than

the exposed surface area of the working electrode in the reagent well;

3. a smaller amount of reagent is needed in the reagent well (4 microliters
of reagent vs. 6 microliters of reagent in the preferred embodiment of Pollmann et

20 al.);

WO 97/02487

PCT/USStf/11240

4. a smaller spreading mesh is needed; and

5. air vents are included on opposing sides of the reagent well.

A smaller sample volume requirement to properly dose the test strip means
fewer underdosing errors will result. This result is especially important for those
persons, such as infants and the elderly who have difficulty in obtaining a reasonably
sized blood drop after pricking their finger with a lancet. The present inventive strip
10 makes it easier for a current measuring meter to discriminate low sample volume
dosing errors. Also, using less reagent per sensor increases production volume for
mass producing sensors. Further, providing side air vents near the reagent well
reduces the occurrence of air bubbles trapped in the reagent well, which results in
fewer testing errors.

15 , : . , ,

The present invention has been disclosed in the above teachings and drawings
with sufficient clarity and conciseness to enable one skilled in the art to make and use
the invention, to know the best mode for carrying out the invention, and to
distinguish it from other inventions and what is old. Many inventions and obvious

20 adaptations of the invention will readily come to mind, and these are intended to be
contained within the scope of the invention as claimed herein.

WO 97/02487

PCT/US96/11240

What is claimed is:

1 . A device for detecting or measuring the concentration of an analyte,
comprising:

a first electrical insulator;

a pair of electrodes consisting of working and counter electrodes of
substantially the same size, the electrodes being made of the same electrically
10 conducting materials and being supported on the first electrical insulator;

a second electrical insulator, overlaying the first electrical insulator and the
electrodes and including a cutout portion that exposes a smaller surface area
of the counter electrode than the working electrode;

a reagent for detecting or measuring the concentration of the analyte, the
reagent substantially covering the exposed electrode surfaces in the cutout
portion; and

20 a spreading mesh, impregnated with a surfactant, overlaying the cutout

portion and affixed to the second electrical insulator,

wherein the cutout portion and spreading mesh are of sufficient size and the
reagent is in sufficient amount to receive a minimum whole blood sample of
25 about 9 microliters for analyzing the analyte.

2. The device of claim 1, wherein the spreading mesh is affixed to the second
substrate by tape having an adhesive on one side and a hole that exposes at
least a portion of the spreading mesh and the cutout portion, and wherein the

30 tape also includes at least one slit near the hole, thereby providing at least one

air vent.

WO 97/02487

PCT/US96/11240

The device of claim 1, farther comprising a current measuring meter in
electrical connection with the working and counter electrodes.

4. The device of claim 2, wherein the tape includes slits on opposing sides of the
5 hole, thereby providing two air vents.

5. The device of claim 2, wherein the cutout portion is 4 millimeters by 4.2
millimeters.

10 6. The device of claim 5, wherein the spreading mesh is 6 millimeters by 5.8
millimeters.

7. The device of claim 6, wherein the amount of reagent is 4 microliters before
drying.

8. The device of claim 7, wherein the spreading mesh is impregnated with
dioctylsodium sulfosuccinate.

9. The device of claim 8, wherein the hole in the tape exposes substantially all of
20 the cutout portion.

10. The device of claim 9, farther comprising a current measuring meter in
electrical connection with the working and counter electrodes.

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US967U240

A. CLASSIFICATION OF SUBJECT MATTER
IPC(6) :GO!N 27/327
USCL :204/403

According to International Patent Classification (IPC) or to both national classification

and IPC

B. FIELDS SEARCHED " "

Minimum documentation searched (clarification system followed by classification symbols)
U.S. : 204/403,416,418,449; 435/817

Documentation searched other than minimum documentation to the extent that such document* are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

CiUtion of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

US, A, 4,842,712 (Seshimoto et al), 27 June 1989, see
column 3, line 30.

2,4-10

US, A, 4,897,173 (Nankai et al), 30 January 1990, see
column 3, lines 26-64.

1-10

US, A, 5,288,636 (Pollmann et al), 22 February 1994, see
column 9, line 43.

1-10

US, A, 5,385,846 (Kuhn et al) 31 January 1995, see
column 2, line 61 to column 3, line 50.

1-10

US, A, 5,395 ,504 (Saurer et al), 07 March 1995, see
column 6, line 61 to column 7, line 16.

1-10

I Further documents arc listed in the continuation of Box C. See

patent family annex.

to be of pvUcukr refcwace

d*te sod ooi ba conflict with the *ppbcadioo bwt cited to u
pnncmle or theory tmdeitymo, die mvcetion

•is

of ptrfacuW tekvmxe; the ckkned htvcnooo cam** be
lend novel or cannot be ennridered to amtvenkmatWasuo
thcdoaaneot»tak«»k»e

r to the

rOtot date but htcrtttto

coeriduwi to involve mi inventive tfep when the
combined with one or more other Rich doc
beat obvioue to ■ pereon akiBod m the art

Date of the actual completion of the international search
20 SEPTEMBER 1996

Date of mailing of the international search report

2 7 NOV 1996

Name and mailing address of the ISA/US
Commueiooer of Patents and Trademark*
BoxPCT

WaabJngtoa, D.C. 20231
Facsimile No. (703) 305-3230

Authorized officer ^ n

T. TUNG r * " t^Si.

Telephone No, (703) 308-3329

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